Antiplasmodial, Antioxidant, and Cytotoxic Activity of Bridelia micrantha a Cameroonian Medicinal Plant Used for the Treatment of Malaria

Introduction Resistance to common antimalarial drugs and persistence of the endemicity of malaria constitute a major public health problem in Cameroon. The aim of this study was to evaluate the in vitro antiplasmodial, antioxidant, and cytotoxic activities of aqueous and ethanol extracts of Bridelia micrantha used by Cameroonian traditional healers for the treatment of malaria. Methods Aqueous and ethanolic stem bark extracts were prepared according to standard procedures. The SYBR Green method was used for antiplasmodial activity on strains of Plasmodium falciparum sensitive to chloroquine (3D7) and resistant (Dd2). In vitro antioxidant activities of B. micrantha were determined using the scavenging activity of 2,2′-diphenyl-1-picrylhydrazyl, nitric oxide, ferric reducing power, and hydrogen peroxide as well as their cytotoxicity on RAW 264.7 macrophage cells and red blood cells (RBC). Results The aqueous and ethanol extracts of Bridelia micrantha showed antiplasmodial activity on the 3D7 strain with IC50 of 31.65 ± 0.79 μg/ml and 19.41 ± 2.93 μg/ml, respectively, as well as 37.64 ± 0.77 μg/ml and 36.22 ± 1.04 μg/ml for the Dd2 strain, respectively. The aqueous and ethanol extracts showed free radical scavenging properties. The IC50 aqueous and ethanol extract was approximately 0.0001737 μg/ml, 42.92 μg/ml, 1197 μg/ml, 63.78 μg/ml and 4.617 μg/ml, 429.9 μg/ml, 511 μg/ml, and 69.32 μg/ml for DPPH, NO, H2O2, and FRAP, respectively, which were compared to ascorbic acid (8.610e − 005 μg/ml, 2901 μg/ml, 3237 μg/ml, and 18.57 μg/ml). The aqueous and ethanol extracts of B. micrantha were found to be nontoxic with CC50 values of 950 ± 6.6 μg/ml and 308.3 ± 45.4 μg/ml, respectively. Haemolysis test showed that the two extracts were not toxic. Conclusion These results suggest that B. micrantha can serve as an antimalarial agent. However, further studies are needed to validate the use of B. micrantha as an antimalarial.

Introduction. Resistance to common antimalarial drugs and persistence of the endemicity of malaria constitute a major public health problem in Cameroon. The aim of this study was to evaluate the in vitro antiplasmodial, antioxidant, and cytotoxic activities of aqueous and ethanol extracts of Bridelia micrantha used by Cameroonian traditional healers for the treatment of malaria. Methods. Aqueous and ethanolic stem bark extracts were prepared according to standard procedures. The SYBR Green method was used for antiplasmodial activity on strains of Plasmodium falciparum sensitive to chloroquine (3D7) and resistant (Dd2). In vitro antioxidant activities of B. micrantha were determined using the scavenging activity of 2,2′-diphenyl-1-picrylhydrazyl, nitric oxide, ferric reducing power, and hydrogen peroxide as well as their cytotoxicity on RAW 264.7 macrophage cells and red blood cells (RBC). Results. The aqueous and ethanol extracts of Bridelia micrantha showed antiplasmodial activity on the 3D7 strain with IC 50 of 31:65 ± 0:79 μg/ml and 19:41 ± 2:93 μg/ml, respectively, as well as 37:64 ± 0:77 μg/ml and 36:22 ± 1:04 μg/ml for the Dd2 strain, respectively. The aqueous and ethanol extracts showed free radical scavenging properties. The IC 50 aqueous and ethanol extract was approximately 0.0001737 μg/ml, 42.92 μg/ml, 1197 μg/ml, 63.78 μg/ml and 4.617 μg/ml, 429.9 μg/ml, 511 μg/ml, and 69.32 μg/ml for DPPH, NO, H2O2, and FRAP, respectively, which were compared to ascorbic acid (8:610e − 005 μg/ml, 2901 μg/ml, 3237 μg/ml, and 18.57 μg/ml). The aqueous and ethanol extracts of B. micrantha were found to be nontoxic with CC 50 values of 950 ± 6:6 μg/ml and 308:3 ± 45:4 μg/ml, respectively. Haemolysis test showed that the two extracts were not toxic. Conclusion. These results suggest that B. micrantha can serve as an antimalarial agent. However, further studies are needed to validate the use of B. micrantha as an antimalarial.

Introduction
Malaria is caused by a protozoan of the genus Plasmodium that is transmitted to humans by the bite of an infected female anopheles mosquito [1]. It is characterised by fever and haemolytic erythrocytopathy due to its presence and development in the liver and red blood cells (RBCs). Malaria is one of the most widespread parasitic diseases globally representing one of the deadliest pandemics for countries in sub-Saharan Africa [2]. It is endemic in the tropical and subtropical areas of Africa, Latin America, and South and South-east Asia, constituting a significant global health burden [3] with about 229 million cases reported in 2021 and over 409,000 deaths. Africa remains the most infected continent, with around 213 million cases recorded [3].
Cameroon is among the 18 countries with 90% of deaths caused by malaria, and 71% of its population is living in areas of high transmission [4]. Children under 5 years and pregnant women are the most infected [5,6]. Malaria is a major threat having a devastating impact on the public health and well-being of Cameroonians [7][8][9]. According to the Ministry of Public Health, malaria is the leading cause of consultation (26%), hospitalization (46%), deaths (22%), and annual household health budgets (40%) [8].
In the absence of an effective vaccine against malaria, chemotherapy, chemoprophylaxis, and vector control remain the principal means of managing malaria. The adoption of artemisinin-based combination therapies (ACTs) as first-line drugs for more than 15 years has led to a reduction in mortality in tropical and subtropical regions. However, this achievement is seriously threatened by decreased clinical efficacy of artemisinins [10], as well as their high cost. Humans have long exploited nature as a source of food and medicine. Plants are thus the oldest and most important healing resource available to man [11]. The WHO estimates that over 20,000 plants are used for the treatment of malaria around the world [12]. Traditional healers in the West Region of Cameroon use Bridelia micrantha to treat malaria, headaches, gastric ulcers, coughs, rheumatism, dysentery, ethnoveterinary medicine, sexually transmitted infections, stomach aches, tapeworms, and diarrhoea [13].
However, the antiplasmodial effect of this plant has not been scientifically evaluated on the different stages of development of P. falciparum.
According to Noumedem et al. [14], there is a direct relationship between malaria and oxidative stress. A plasmodial infection leads to the overproduction of reactive oxygen species. These free radicals are not only toxic to the parasite; they are equally toxic to the host organism. Hence, an antiplasmodial remedy with good antioxidant properties will be an advantage. This study therefore reports the antiplasmodial and antioxidant activities of B. micrantha used by traditional healers in the Western Region of Cameroon to treat malaria.

Material and Methods
2.1. Collection and Identification. Bridelia micrantha's stem bark was collected in 2021 in Foumban, West Region of Cam-eroon. The leaves, seeds, and flowers were sent to the National Herbarium of Cameroon for identification with reference number 64129/HNC attributed to the voucher specimen.

Preparation of Plant
Extracts. Powder B. micrantha (100 grams) using an electrical balance (SF-400) was weighed into a 2-litre container, with an addition of 95% ethanol (one litre) followed by agitation for five minutes and maceration for 72 hours. Whatman paper no. 3 was used to filter the homogenate with the resulting filtrate evaporated at 45°C in an oven [15]. The aqueous extract was prepared by infusion in distilled water at 100°C for three hours, followed by filtration to obtain the filtrate.

2.3.2.
Synchronization of the Culture. The parasite cultures that included a majority of the ring stage (>80%) were synchronized at the ring stage before testing antiplasmodial activity. This was done by treating them with 5% (w/v) sorbitol for 10 minutes [17].

In Vitro Test for P. falciparum Growth Inhibition Based on Present Full Meaning of the Acronym (SYBR) Green
Fluorescence. The in vitro antiplasmodial activity was evaluated according to the method described by Smilkstein et al. [18]. Briefly, 10 μl of the various concentrations of extracts, artemisinin, and chloroquine were put in contact into a 96-well microplate titer with 90 μl of the parasite suspension at the ring stage of 2% parasitemia and 1% hematocrit. The plates were then incubated for 72 hours at 37°C in a CO 2 incubator. The final plant extract concentration ranged from 0.01258 to 200 μg/ml. This experiment was done in triplicates. One hundred microlitres (100 μl) of SYBR Green was added into each well followed by an hour of incubation in the darkness. The result of the antiplasmodial activity was read using an ELISA fluorescence microplate reader (Tecan Infinite M200) at an excitation and emission wavelength of 485 and 538 nm, respectively. The resistance index (RI) was calculated using the formula: 2.4. In Vitro Antioxidant Activity of B. micrantha 2.4.1. DPPH Antiradical Activity. The antifree radical activity will be measured using DPPH by the method of [19].
In the spectrophotometer cuvettes, 0.5 ml of plant or vitamin C extracts (at different final concentrations: 1, 3, 10, 30, 100, and 300 μg/ml) will be mixed with 0.5 ml of methanol and 0.5 ml of DPPH (0.063 mg/ml). The blank will consist of 1 ml of methanol and 0.5 ml of DPPH. The absorbance will be read at 517 nm before (DOi) and 20 minutes (DOf) after the introduction of the DPPH solution into the reaction medium, respectively. Vitamin C will serve as a positive control and the test will be performed in 3 replicates.
2.4.2. Ferric Reducing Power. The procedure described by Bokhari et al. [20] was followed with slight modification. Briefly, 200 μl of plant extracts/vitamin C were added to test tubes together with 0.5 ml of phosphate-buffered solution (200 mM, pH 6.6) and 0.5 ml of potassium ferricyanide solution. The final concentrations of the plant extracts and vitamin C were 1, 3, 10, 30, 100, and 300 g/ml (30 mM). The absorbance was measured at 700 nm, and the mixture will be incubated for 10 minutes of incubation at 37°C. The test was carried out in triplicate.

Nitric Oxide (NO) Inhibition Test (IT).
When oxygen and NO are combined in vitro at physiological pH, they create nitrite ions which may be detected by the Griess reaction [21]. Ten milligrams (10 mg) of extract and vitamin C (at a concentration of 1, 3, 10, 30, 100, or 300 g/ml) were dissolved in 3.53 ml of phosphate-buffered saline (pH = 7:4, 10 mH, pKa = 6:9) to obtain a stock solution. Briefly, 1520 μl of sodium nitroprusside (10 mM) was introduced into test tubes containing 180 μl of Aframomum pruinosum or vitamin C extract. The mixture was incubated at 25°C for 2 h 30 min. At the end of the incubation, 500 μl of the previous mixture was taken and introduced into spectrophotometer cuvettes, and then, an equivalent volume of 1% sulfanilamide was added. The mixture was homogenized and incubated for 5 min at room temperature in the dark. 500 μl of naphthyl ethylenediamine (NED, 0.1%) was added to the mixture, followed by another 5 minutes of incubation in the darkness. The absorbance was read at 530 nm to measure the production of the chromophore. The scavenging activity was calculated as follows:  [23]. Five hundred microlitres (500 μl) of a suspension of healthy erythrocytes from fresh O + blood was prepared at 4% hematocrit in incomplete RPMI1640, in the presence of 500 μl plant extracts at different concentrations in the Eppendorf tubes. Under the same conditions, Triton X-100 at 0.5% (for 100% haemolysis) and the erythrocyte suspension in an incomplete culture medium at 4% hematocrit were used as positive control and negative control, respectively. The final concentrations in the test plates varied from 1000 to 62.5 μg/ml (0.5% DMSO) for the extracts and 0.5% for the Triton X-100 in a final volume of 1000 μl. The plates were incubated at 37°C for 3 hours. After incubation followed by centrifugation at 2500 rpm/3 min, the absorbance of the supernatant corresponding to the release of hemoglobin was measured at 540 nm using the Infinite M200 microplate reader (Tecan). The haemolysis rate of the various extracts was estimated using the formula: where DO sample is the absorbance of sample, DO negative control is the absorbance of negative control, and DO positive control is the absorbance of positive control.

Cytotoxicity
Test on RAW 264.7 Cells. The resazurinbased assay [24] was used to test the extract's ability to kill RAW 264.7 cells. Macrophages were seeded at a density of 10 4 cells per 100 μl of complete media. After a 24-hour incubation at 37°C with 5% CO 2 , test plates were filled with 10 μl of each serially diluted test sample solution (extract) and then incubated for another 48 hours. Ten microlitres of 0.15 mg/ml of resazurin was added to each well and incubated for 4 hours. Cell proliferation was assessed by measuring the absorbance at 540 nm using the plate reader (Tecan Infinite M200). The selectivity index (SI) was calculated according to the following formula: For this purpose, the regression lines were drawn using the values read from the different inhibition percentages and the decimal logarithm of the extract concentrations ½% inhibition = f ðlog CÞ. The equations of the regression lines of the form y = ax + b were used. Assuming each time that y = 50, we obtain IC 50 and CC 50 = 10x, where x = ð50 − bÞ/a. Microsoft Excel software. Next, the replicate number averages of the 50% inhibitory concentration (IC 50 ) were determined using concentration-response curves obtained by plotting the logarithm of the concentration versus the percentage inhibition using the GraphPad Prism 8 software. P < 0:05 was significant. Table 1 shows the IC 50 of the aqueous and ethanol extracts of B. micrantha on chloroquine-resistant (Dd2) and chloroquine-sensitive (3D7) strains. It appears from this table that the ethanol extract was more active than the aqueous extract on the Dd2 and 3D7 strains, with IC 50 values of 36:22 ± 1:04 g/ml and 19:41 ± 2:93 g/ml, respectively. The aqueous and ethanol extracts had resistance indexes (RI) of 1.18 and 1.9, respectively.

Antioxidant Activity of B. micrantha
3.2.1. DPPH Scavenging Activity. Figure 1 shows the scavenging activity of B. micrantha. The aqueous and ethanol extract have inhibited the production of a DPPH radical. Ascorbic acid showed an IC 50 of 8:610e − 005, whereas the aqueous and ethanol extracts had values of 0.0001737 μg/ ml and 4.617 μg/ml, respectively.

Nitric Oxide Free Radical Inhibition.
The results obtained from the NO production inhibition test are represented in Figure 2. This figure shows that the aqueous and ethanol extracts decrease the production of NO with an increase in the concentration of the extracts. The IC 50 values of the aqueous extract, ethanolic extract, and ascorbic acid were 42.92, 429.9, and 2901 μg/ml, respectively. Figure 3 shows the results obtained for the hydrogen peroxide test of the extracts. The results obtained showed that the standard and extract absorbance increased with respect to concentrations. It emerges from this figure that the aqueous and ethanol extracts inhibited the production of hydrogen peroxide more than ascorbic acid. The IC 50 of ascorbic acid was 3237 μg/ml which was higher than that of the aqueous and ethanol extracts, with IC 50 of 1197 and 511 μg/ml, respectively.

Haemolysis
Test. The haemolytic activity of plant extracts is expressed as a percentage of haemolysis. The two samples exhibited a very weak haemolytic effect against human erythrocytes. However, these extracts showed a dosedependent increase in haemolytic activity ( Figure 5). The concentrations of 1000 μg/ml and 62.5 μg/ml had an inhibition percentage of 18.86% and 1.52%, respectively.

Discussion
The results of our investigation showed that the IC 50 values of the aqueous and ethanolic extracts were 36:22 ± 1:04 μg/ml and 19:41 ± 2:93 μg/ml and 37:64 ± 0:77 μg/ml and 31:65 ± 0:79 μg/ml, respectively, for susceptible Pf3D7 and resistant PfDd2 strains. According to the classification of Kumari et al. [25], when the IC 50 is higher than 5 μg/ml, the extract is considered very active; when it is between 5 and 50 μg/ ml, it is active; when it is between 50 and 100 μg/ml, its activity is moderate; and when it is higher than 100 μg/ml, it is inactive. We can conclude that our aqueous and ethanolic extracts are active on susceptible Pf3D7 and resistant PfDd2 strains. In Nigeria, a similar study was conducted, and the IC 50 for B. micrantha was 158:7 ± 7:82 g/ml [26]. The IC 50 obtained by Ramadhani et al. [27] was 8 g/ml for B. micrantha decoction. The difference in IC 50 observed may be due to the fact that different strains of Plasmodium were used to conduct the experiment. It appears from this study that the ethanolic extract was more active than the aqueous extract. Our results are similar to those obtained by Tomani et al. [28] with Eriosema montanum Baker f. roots on Plasmodium 3D7 strain which had obtained an IC 50 of 17:68 ± 4:030 μg/ml of ethanolic extract and different from that obtained by Esseh et al. [29] which had an IC 50 of 91:08 ± 0:61 μg/ml of aqueous extract. This difference can be explained by the type of extract, and the strain of

BioMed Research International
Plasmodium used in this study, as well as the geographical location of the plant, may have had an impact on the observed difference. The resistance index (RI) indicates the inhibitory potential of a drug against sensitive and resistant strains of P. falciparum. The resistance index was in the range of 1.18 to 1.9. Extract with RI < 1 is considered promising against sensitive and resistant strains. This shows that the extract of B. micrantha had good resistance indices and was classified as interesting and promising.
Bridelia micrantha extracts demonstrated strong DPPH free radical scavenging efficacy implying that they possess robust antioxidant properties. The IC 50 values of the extracts were 0.00017 μg/ml and 4.617 μg/ml for the aqueous and ethanol. A similar study carried out on Euphorbia neriifolia of the same family had an IC 50 of 76:2 ± 0:07 inhibition [30]. This could be justified by the fact that all plants belonging to the same family do not necessarily have the same mode of action. Bridelia micrantha extract has the ability to donate protons that can serve as an inhibitor or scavenger of free radicals, acting as a primary [30].
These plant extracts increased the amount of nitrite produced from the breakdown of sodium nitroprusside in vitro, suggesting that direct scavenging of the NO radical may not be related to the suppression of its release. The plant extract did not significantly improve the dose-dependent scavenging of nitric oxide. The IC 50 values for the extracts were 42.92 μg/ml for the aqueous and 429.9 μg/ml for the ethanol extract, while the IC 50 value of ascorbic acid was 2901 μg/ml. This result suggests that B. micrantha extracts would not intercept the NO formation reaction from nitroprusside but would rather act in favor of this reaction. On the other hand, these extracts could be beneficial in the management of arterial hypertension, one of the major characteristics of which is the decrease in the bioavailability of NO. Nitric oxide and superoxide anions have the potential to injure a variety of tissues. Peroxynitrite (ONOO -), which is formed when NO and O 2react, worsens the toxicity profile and damages it because it causes toxic interactions with biomol-ecules [31,32]. The cascade of reactions triggered by excessive NO formation can be stopped by scavenging the reactive peroxynitrite, which will help stop the negative effects that these radicals have in the human body.
The H 2 O 2 scavenging activity of the aqueous and ethanolic extracts was slightly higher than the positive control. The IC 50 values of the extract were 1197 μg/ml and 511 μg/ml for the aqueous and ethanol, while the IC 50 value of ascorbic acid was 3237 μg/ml. Hydrogen peroxide can produce hydroxyl ions in the presence of iron ions, which can be harmful to cells [33]. Therefore, in order to provide antioxidant defense, H 2 O 2 must be removed from cells. The reducing power of B. micrantha extracts increases as the concentration increases. This is so because reducers break the chain of the free radicals donating a hydrogen atom. Their presence is closely related to the reducing activities of extracts [34]. Since a considerable reducing activity was noticed, it means our plant extracts contain phytochemical constituents capable of reacting with ferric-tripyridyl triazine (Fe 3+ -TPTZ) complex to produce Fe 2+ -TPTZ as the end product.
The CC 50 values of the extracts in this study were 950 ± 6:6 μg/ml and 308:3 ± 45:4 μg/ml for the aqueous and ethanol extracts, respectively, on RAW 264.7 macrophage cells. According to the cytotoxicity classification of Malebo et al., [35] a CC 50 < 1:0 μg/ml is high cytotoxicity, CC 50 < 1:0 -10:0 μg/ml is moderate cytotoxicity, CC 50 < 10:0 − 30 μg/ml is average cytotoxicity, and CC 50 > 30:0 μg/ml is noncytotoxic. Hence, the extracts from B. micrantha can be classified as nontoxic. The aqueous extract had a SI of 30.01 and 25.23 for Pf3D7 and PfDd2, respectively, and was higher than that of the ethanol extract which was 15.9 and 8.51 for Pf3D7 and PfDd2, respectively. A similar study was conducted by Bapela et al. [36] on B. mollis Hutch where they had a CC 50 of 51.4 μg/ml with a SI of 17. The difference observed here may be due to the cell strain used for the cytotoxicity test.
Many researchers have used erythrocytes as a model system to explore the interaction of drugs with membranes 6 BioMed Research International [37]. The concentration and potency of the extract are linked to haemolysis. In addition, the chemical composition of each extract affects its haemolytic action. The aqueous and ethanol extracts of B. micrantha had no effect and does not affect the erythrocyte membrane at low concentrations, but attention should be given to high doses like 1000 μg/ml.

Conclusion
The aqueous and ethanol extracts of B. micrantha were found to be active against Plasmodium falciparum-sensitive 3D7 and -resistant Dd2 strains but nontoxic on RBCs and macrophage cells (RAW 264.7). The results also showed that the aqueous and ethanol extracts of B. micrantha have promising antioxidant activity, suggesting that it may be useful in the prevention of many oxidative stress-related diseases. However, studies must be carried out to scientifically confirm the use of B. micrantha as an antiplasmodial remedy and to assess its safety.

Data Availability
All data generated and analyzed are included in this research article.

Conflicts of Interest
The authors declare that there is no conflict of interest.